The as-yet-uncultured bacteria are a rich potential resource for natural product discovery. In most environments, the readily cultured bacteria represent <1% of the total community, and soil is likely to be one of the most biologically diverse, as it is home to 4,000 to 40,000 species/g. Soil's biological diversity is matched by its chemical diversity: soil has been the source of more antibiotics and other medicinal compounds than any other natural habitat, but virtually all of the chemistry is derived from the readily cultured members of the soil community. We have developed functional metagenomics, the genomic analysis of assemblages of bacteria, as a means to access the biological and chemical potential of soil microbial communities. Functional metagenomics entails extracting DNA directly from soil, cloning it into a bacterial host, and screening the resulting libraries for activities of interest. The major limitation in functional metagenomic analysis to date has been the low frequency of active clones among libraries with many clones. The low frequency is likely due to (1) low expression of foreign genes in the bacteria harboring the soil DNA and (2) insensitivity of current screens. In the proposed research, we aim to develop universal strategies to address both of these limitations that will be broadly applicable to functional metagenomic analysis of any environment for diverse functions. We intend to develop expression systems in representatives of four diverse divisions of bacteria: Gamma-Proteobacteria (E. coli), alpha-Proteobacteria (Agrobacterium tumefaciens), Firmicutes (Bacillus subtilis), and Actinobacteria (Streptomyces coelicolor). To screen libraries maintained in each of these hosts, we will design and implement intracellular screens in which the metagenomic DNA is inside the same cell as a biosensor that detects diverse natural products. These screens are sensitive and rapid and can be applied easily to libraries containing millions of clones. This research will lead to universal expression systems that will be useful for metagenomic analysis and other techniques that rely on heterologous gene expression.